Mechanism for autoinhibition and activation of the MORC3 ATPase.

Microrchidia 3 (MORC3) is a human protein linked to autoimmune disorders, Down syndrome, and cancer. It is a member of a newly identified family of human ATPases with an uncharacterized mechanism of action. Here, we elucidate the molecular basis for inhibition and activation of MORC3. The crystal structure of the MORC3 region encompassing the ATPase and CW domains in complex with a nonhydrolyzable ATP analog demonstrates that the two domains are directly coupled. The extensive ATPase:CW interface stabilizes the protein fold but inhibits the catalytic activity of MORC3. Enzymatic, NMR, mutational, and biochemical analyses show that in the autoinhibited, off state, the CW domain sterically impedes binding of the ATPase domain to DNA, which in turn is required for the catalytic activity. MORC3 autoinhibition is released by disrupting the intramolecular ATPase:CW coupling through the competitive interaction of CW with histone H3 tail or by mutating the interfacial residues. Binding of CW to H3 leads to a marked rearrangement in the ATPase-CW cassette, which frees the DNA-binding site in active MORC3 (on state). We show that ATP-induced dimerization of the ATPase domain is strictly required for the catalytic activity and that the dimeric form of ATPase-CW might cooperatively bind to dsDNA. Together, our findings uncovered a mechanism underlying the fine-tuned regulation of the catalytic domain of MORC3 by the epigenetic reader, CW.

The nucleosome position-encoding WW/SS sequence pattern is depleted in mammalian genes relative to other eukaryotes.

Nucleosomal DNA sequences generally follow a well-known pattern with ∼10-bp periodic WW (where W is A or T) dinucleotides that oscillate in phase with each other and out of phase with SS (where S is G or C) dinucleotides. However, nucleosomes with other DNA patterns have not been systematically analyzed. Here, we focus on an opposite pattern, namely anti-WW/SS pattern, in which WW dinucleotides preferentially occur at DNA sites that bend into major grooves and SS (where S is G or C) dinucleotides are often found at sites that bend into minor grooves. Nucleosomes with the anti-WW/SS pattern are widespread and exhibit a species- and context-specific distribution in eukaryotic genomes. Unlike non-mammals (yeast, nematode and fly), there is a positive correlation between the enrichment of anti-WW/SS nucleosomes and RNA Pol II transcriptional levels in mammals (mouse and human). Interestingly, such enrichment is not due to underlying DNA sequence. In addition, chromatin remodeling complexes have an impact on the abundance but not on the distribution of anti-WW/SS nucleosomes in yeast. Our data reveal distinct roles of cis- and trans-acting factors in the rotational positioning of nucleosomes between non-mammals and mammals. Implications of the anti-WW/SS sequence pattern for RNA Pol II transcription are discussed.

PKCδ Regulates Chromatin Remodeling and DNA Repair through SIRT6.

Irradiation (IR) is a highly effective cancer therapy; however, IR damage to tumor-adjacent healthy tissues can result in significant comorbidities and potentially limit the course of therapy. We have previously shown that protein kinase C delta (PKCδ) is required for IR-induced apoptosis and that inhibition of PKCδ activity provides radioprotection in vivo. Here we show that PKCδ regulates histone modification, chromatin accessibility, and double-stranded break (DSB) repair through a mechanism that requires Sirtuin 6 (SIRT6). Overexpression of PKCδ promotes genomic instability and increases DNA damage and apoptosis. Conversely, depletion of PKCδ increases DNA repair via nonhomologous end joining (NHEJ) and homologous recombination (HR) as evidenced by increased formation of DNA damage foci, increased expression of DNA repair proteins, and increased repair of NHEJ and HR fluorescent reporter constructs. Nuclease sensitivity indicates that PKCδ depletion is associated with more open chromatin, while overexpression of PKCδ reduces chromatin accessibility. Epiproteome analysis reveals increased chromatin associated H3K36me2 in PKCδ-depleted cells which is accompanied by chromatin disassociation of KDM2A. We identify SIRT6 as a downstream mediator of PKCδ. PKCδ-depleted cells have increased SIRT6 expression, and depletion of SIRT6 reverses changes in chromatin accessibility, histone modification and DSB repair in PKCδ-depleted cells. Furthermore, depletion of SIRT6 reverses radioprotection in PKCδ-depleted cells. Our studies describe a novel pathway whereby PKCδ orchestrates SIRT6-dependent changes in chromatin accessibility to regulate DNA repair, and define a mechanism for regulation of radiation-induced apoptosis by PKCδ. IMPLICATIONS: PKCδ controls sensitivity to irradiation by regulating DNA repair.

Genomic Redistribution of Metal-Response Transcription Factor-1 (MTF-1) in Cadmium Resistant Cells.

(1) Background: Metal homeostasis is an important part of cellular programs and is disrupted when cells are exposed to carcinogenic heavy metals. Metal response is mediated by the metal response element transcription factor MTF-1. However, where MTF-1 binds and how that binding changes in response to heavy metals, such as cadmium, remains unknown. (2) Methods: To investigate the effects of prolonged cadmium exposure on the genomic distribution of MTF-1, we performed MTF-1 CUT&RUN, RNA-seq and ATAC-seq on control and cadmium-resistant cells. (3) Results: Changes in MTF-1 binding primarily occur distal to the transcription start sight. Newly occupied MTF-1 sites are enriched for FOS/JUN DNA binding motifs, while regions that lose MTF-1 binding in cadmium are enriched for the FOX transcription factor family member DNA binding sites. (4) Conclusions: Relocalization of MTF-1 to new genomic loci does not alter the accessibility of these locations. Our results support a model whereby MTF-1 is relocalized to accessible FOS/JUN-bound genomic locations in response to cadmium.

Transition from Transient DNA Rereplication to Inherited Gene Amplification Following Prolonged Environmental Stress.

Cells require the ability to adapt to changing environmental conditions, however, it is unclear how these changes elicit stable permanent changes in genomes. We demonstrate that, in response to environmental metal exposure, the metallothionein (MT) locus undergoes DNA rereplication generating transient site-specific gene amplifications (TSSGs). Chronic metal exposure allows transition from MT TSSG to inherited MT gene amplification through homologous recombination within and outside of the MT locus. DNA rereplication of the MT locus is suppressed by H3K27me3 and EZH2. Long-term ablation of EZH2 activity eventually leads to integration and inheritance of MT gene amplifications without the selective pressure of metal exposure. The rereplication and inheritance of MT gene amplification is an evolutionarily conserved response to environmental metal from yeast to human. Our results describe a new paradigm for adaptation to environmental stress where targeted, transient DNA rereplication precedes stable inherited gene amplification.

PKCδ regulates chromatin remodeling and DNA repair through SIRT6.

Protein kinase C delta (PKCδ) is a ubiquitous kinase whose function is defined in part by localization to specific cellular compartments. Nuclear PKCδ is both necessary and sufficient for IR-induced apoptosis, while inhibition of PKCδ activity provides radioprotection in vivo. How nuclear PKCδ regulates DNA-damage induced cell death is poorly understood. Here we show that PKCδ regulates histone modification, chromatin accessibility, and double stranded break (DSB) repair through a mechanism that requires SIRT6. Overexpression of PKCδ promotes genomic instability and increases DNA damage and apoptosis. Conversely, depletion of PKCδ increases DNA repair via non-homologous end joining (NHEJ) and homologous recombination (HR) as evidenced by more rapid formation of NHEJ (DNA-PK) and HR (Rad51) DNA damage foci, increased expression of repair proteins, and increased repair of NHEJ and HR fluorescent reporter constructs. Nuclease sensitivity indicates that PKCδ depletion is associated with more open chromatin, while overexpression of PKCδ reduces chromatin accessibility. Epiproteome analysis revealed that PKCδ depletion increases chromatin associated H3K36me2, and reduces ribosylation of KDM2A and chromatin bound KDM2A. We identify SIRT6 as a downstream mediator of PKCδ. PKCδ-depleted cells have increased expression of SIRT6, and depletion of SIRT6 reverses the changes in chromatin accessibility, histone modification and NHEJ and HR DNA repair seen with PKCδ-depletion. Furthermore, depletion of SIRT6 reverses radioprotection in PKCδ-depleted cells. Our studies describe a novel pathway whereby PKCδ orchestrates SIRT6-dependent changes in chromatin accessibility to increase DNA repair, and define a mechanism for regulation of radiation-induced apoptosis by PKCδ.

Molecular mechanism of the MORC4 ATPase activation.

Human Microrchidia 4 (MORC4) is associated with acute and chronic pancreatitis, inflammatory disorders and cancer but it remains largely uncharacterized. Here, we describe the structure-function relationship of MORC4 and define the molecular mechanism for MORC4 activation. Enzymatic and binding assays reveal that MORC4 has ATPase activity, which is dependent on DNA-binding functions of both the ATPase domain and CW domain of MORC4. The crystal structure of the ATPaseCW cassette of MORC4 and mutagenesis studies show that the DNA-binding site and the histone/ATPase binding site of CW are located on the opposite sides of the domain. The ATPase and CW domains cooperate in binding of MORC4 to the nucleosome core particle (NCP), enhancing the DNA wrapping around the histone core and impeding binding of DNA-associated proteins, such as transcription factors, to the NCP. In cells, MORC4 mediates formation of nuclear bodies in the nucleus and has a role in the progression of S-phase of the cell cycle, and both these functions require CW and catalytic activity of MORC4. Our findings highlight the mechanism for MORC4 activation, which is distinctly different from the mechanisms of action observed in other MORC family members.

MORC3 Forms Nuclear Condensates through Phase Separation.

Phase separation can produce local structures with specific functionality in the cell, and in the nucleus, this can lead to chromatin reorganization. Microrchidia 3 (MORC3) is a human ATPase that has been implicated in autoimmune disorders and cancer. Here, we show that MORC3 forms phase-separated condensates with liquid-like properties in the cell nucleus. Fluorescence live-cell imaging reveals that the MORC3 condensates are heterogeneous and undergo dynamic morphological changes during the cell cycle. The ATPase activity of MORC3 drives its phase separation in vitro and requires DNA binding and releasing the MORC3 CW domain-dependent autoinhibition through association with histone H3. Our findings suggest a mechanism by which the ATPase function of MORC3 mediates MORC3 nuclear compartmentalization.